Feruloyl oligosaccharides, isolated from bacterial fermented wheat bran, exhibit antioxidant effects in IPEC‐J2 cells and zebrafish model

Abstract Feruloyl oligosaccharides (FOs) were produced by solid‐state fermentation of wheat bran using Bacillus subtilis, Bacillus licheniformis, and Saccharomyces cerevisiae, and its antioxidant activity was investigated using IPEC‐J2 cells and zebrafish embryo model. Preliminary structure analysis revealed that FOs has an average molecular weight of 11.81 kDa and consists of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose. The obtained FOs possess superior reducing power and DPPH and hydroxyl free radical scavenging activities. In IPEC‐J2 cells, antioxidant enzymes activities and GSH level were significantly increased, while MDA level was reduced by FOs. Further studies showed that FOs achieved the aforementioned effects by activating Nrf2 signaling pathway. In zebrafish embryo, FOs effectively suppressed ROS production, lipid peroxidation, and cell death by increasing SOD and GSH‐Px activities. Our findings suggested that FOs from solid‐state fermented wheat bran with mixed bacteria can be used as an antioxidant food additive or drugs.

. Thus, antioxidants play an important role in the maintenance of organism health through modulating redox homeostasis. The identification of effective antioxidants to decrease oxidative stress has become an increasingly important field of research Xie et al., 2020).
Feruloyl oligosaccharides (FOs), which are found in many cereals, are formed by an oligosaccharide ester linked to one or multiple ferulic acid units (Tamayo & Karboune, 2019). As functional oligosaccharides, FOs can eliminate DPPH and hydroxyl free radicals.
FOs have been shown to enhance antioxidant enzyme activity and decrease oxidized glutathione and malondialdehyde levels in plasma of rats (Wang et al., 2010). Previous study has indicated that FOs exhibit higher antioxidant activity than free FA due to the existence of unique ester bonds in its structure (Ou et al., 2007). However, the further development of FOs as a functional food additive has been hindered due to the inefficiency of its production. Therefore, many different methods of producing FOs have been tested, including high-pressure hydrothermal treatment (Yang et al., 2015), trifluoroacetic acid hydrolysis (Li et al., 2008), and xylanases hydrolyses (Katapodis et al., 2003). Furthermore, FOs can be obtained from wheat bran by 7 days of deep fermentation by Agrocybe chaxingu, however, the operation is tedious and time-consuming. Preparation of the inoculum requires incubating A. chaxingu on potato dextrose agar plate at 25°C for 7 days and then transferring to liquid culture of potato dextrose for another 5 days (Xie et al., 2014). Recently, it was discovered that bacterial fermentation is advantageous due to its characteristics of short time consumption and easy operation. Ferreira et al. found that FOs were released from beet meal by 24 h fermentation using Streptomyces tendae (Ferreira et al., 2007).
However, few information is accessible regarding the application of mixed bacterial fermentation in FOs release. Given this gap in our understanding, we used B. subtilis, B. licheniformis, and S. cerevisiae to ferment wheat bran to obtain FOs. It was known that the mixed bacterial fermentation could increase the yield of FOs Chen, Wang, Yin, et al., 2021), but the antioxidant activity of the obtained FOs has not yet been investigated.
IPEC-J2 cells and zebrafish (Danio rerio) are rapid and convenient model systems for the study of bioactivity of natural substances Pan et al., 2013;Zhao et al., 2020). Zebrafish has special advantages over other experimental animal models, due to their smaller size, high breeding capacity, and short life cycle. The morphological and molecular basis of the tissues and organs in the zebrafish are either identical or like humans, respectively. Thus, it is listed as the third largest model organism after human beings and mice. Recently, zebrafish have been used to evaluate the antioxidant activity of natural products (Kim et al., 2014). Therefore, the objective of this study was to determine whether FOs produced from wheat bran by fermentation using B. subtilis, B.
licheniformis, and S. cerevisiae have antioxidant activity. The IPEC-J2 and zebrafish embryo were used as in vitro cell model and in vivo model, respectively. We further evaluated whether the Nrf2 signaling pathway might be involved.

| Fermentation of wheat bran and isolation of FOs
The fermentation of wheat bran and isolation of FOs were conducted according to previous studies Chen, Wang, Yin, et al., 2021). Wheat bran was crushed, screened, and sterilized for 90 min at 121°C. The pasteurized wheat bran was mixed with sterile distilled water at a ratio of 1:1.16 as the fermentation substrate, and inoculated with activated B. subtilis (CGMCC 1.892), B. licheniformis (CGMCC 1.813), and S. cerevisiae (CGMCC 2.119) at a ratio of 1:1:1. Fermentation was conducted at 42.5°C for 58.5 h.
After fermentation, the substrate was dried at 45°C for 48 h, extracted with deionized water (1:10, w/v) at 80°C for 30 min, and centrifuged at 4000 g for 10 min to obtain the fermented wheat bran extract. The extract was mixed with 80% ethanol at 4°C for a night. The crude FOs were collected by centrifugation at 4000 g for 10 min and then lyophilized. The crude FOs (160 mg) were dissolved in 40 ml of distilled water, then pH value of the solution was adjusted to 4. The solution was separated by an Amberlite XAD-2 column (2.6 × 30 cm 2 ) with 50% ethanol solution at a flow rate of 2 ml per min. The eluent obtained from the previous step was freeze-dried, dissolved by distilled water (1:100, w/v), and further purified by a Sephadex LH-20 column (2.6 × 30 cm 2 ) and was eluted with 25% ethanol at a flow rate of 0.3 ml/min to obtain FOs after freeze drying.

| Characterization of FOs
Ferulic acid (FA) was quantified according to a procedure adapted from Rondini et al. (2004). The obtained FOs (0.5 mg/ml) were analyzed using a UV-vis spectrophotometer (UV-1800;Shimadzu) in the range of 200-400 nm. The monosaccharide composition was measured by high-performance gel permeation chromatography (HPGPC) with PMP precolumn derivatization and the molecular weight was measured by gel permeation chromatography (GPC) as mentioned in our earlier study .

| Reducing power
The reducing power of FOs was determined according to Xiao et al.'s method (2021). Briefly, 0.75 ml of FOs solution in a series of concentrations (from 0.5 to 4.0 mg/ml) was mixed with 0.75 ml phosphate buffer (0.2 mol/L, pH 6.6) and 0.75 ml 1% potassium ferricyanide (K 3 Fe[CN] 6 ). The mixture was incubated at 50°C for 20 min, then 0.75 ml of 10% trichloroacetic acid (TCA) was added to stop the reaction. The mixture was centrifuged at 4500 g for 10 min.
The supernatant (1.5 ml) was mixed with 1.5 ml distilled water and 400 μl 0.1% FeCl 3 . After incubation at room temperature for 10 min, the absorbance of the mixture was measured at 700 nm.
The mixture was shaken and incubated at room temperature in the dark for 30 min, and the absorbance was measured at 517 nm against a blank (water instead of sample solution). The DPPH radical scavenging activity was calculated using the following equation: where A 0 is the absorbance of the blank, A 1 is the absorbance of a mixture of the sample and DPPH solutions, and A 2 is the absorbance of the sample solution without DPPH.

| Hydroxyl radical scavenging activities
The scavenging activity against hydroxyl radical of FOs was measured based on the assay reported of .
After incubation at 37°C for 30 min, the absorbance at 510 nm was measured. The hydroxyl radical scavenging activity was calculated using the following equation: where A a is the absorbance of the resulting mixture without sample solution, A b is the absorbance of the resulting mixture with the sample, and A c is the absorbance of the resulting mixture without FeSO 4 .

| Intracellular ROS assays
Intracellular ROS generation in IPEC-J2 cells was determined using a reactive oxygen species assay kit (Nanjing Jiancheng Bioengineering Institute) according with the manufacturer's instructions. Briefly, cells were seeded with different concentrations of FOs (0, control; 50, 100, 200, 400 μg/ml) for 24 h of exposure in a humidified incubator (5% CO 2 , 37°C). Thereafter, the cells were stained for 20 min with DCFH-DA staining solution, rinsed in serum-free medium for three times, air-dried, and examined under an optical microscope (TS2R, Nikon, Japan) equipped with a digital camera.

| Antioxidant indices assays
The activities of CAT, SOD, and GSH-Px and levels of GSH and MDA were determined using colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute, China). Samples were measured according with the manufacturer's instructions. The content of total protein was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce).

| Real-time PCR assays
Total RNA was extracted using TRNzol Universal reagent (Tiangen Biotech Co., Ltd.), and 1 μg of RNA was reverse transcribed using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara Bio).
Real-time PCR was performed using a ABI7500 system (Applied The day before an experiment, males (10) were bred with females (5) to obtain embryos. Natural spawning was stimulated with lights on in the morning to obtain the embryos, and collection of embryos was completed within 30 min.

| Toxicity of FOs in zebrafish embryos
The embryos (n = 10) were transferred to individual wells of 24-well plates with 2 ml embryo media at 7-9 h post fertilization (hpf). The embryos were treated with FOs (0, 50, 100, 200, 400, or 800 μg/ ml). The survival rate of zebrafish embryos after 72 h of exposure was calculated.

| Effect of FOs on zebrafish embryos
Approximately 7-9 hpf, the embryos (10 embryos/well, 28 wells/ The ROS production, lipid peroxidation, and cell death of zebrafish embryos were detected using an DCF-DA, DPPP, and acridine orange, respectively. The fluorescence intensity of individual zebrafish larvae was quantified using ImageJ software . The CAT, SOD, and GSH-Px activity were also measured using the commercial assay kits from Nanjing Jiancheng Bioengineering Institute.

| Statistical analysis
The experiments were performed in triplicate, and the data were expressed as the mean ± standard error (SE). One-way analysis of variance (ANOVA) by SAS statistical software. Significant differences between the means were identified by the Tukey's test. The diversity between groups was indicated at statistically dramatic level of p < .05.

| Preliminary structure of FOs
Feruloyl oligosaccharides were prepared from fermented wheat bran and purification with macroporous resin Amberlite XAD-2 and Sephadex LH-20. As shown in Figure 1, the UV absorption spectra suggested that the FOs had an absorption wavelength of 286 and 325 nm, and a marker of esterified feruloyl group .

| In vitro antioxidant activities of FOs
Reducing power is often used to evaluate the ability of an antioxidant to donate an electron (Hamed et al., 2020). Reducing power and absorbance values were correlated positively. Figure 3a shows the reducing power of FOs at different concentrations. FOs showed a weak degree of electron donation capacity in the range of 0.5-2.0 mg/ml.
FOs exhibited a significantly lower absorbance value when compared to BHA (p < .05). However, FOs at a concentration of 4.0 mg/ml exhibited excellent reducing power (1.97), which was higher than that of BHA (1.80), but not significantly different (p > .05).
The DPPH free radical is a stable nitrogen-centered free radical with an unpaired electron, which will transform into stable form by taking a hydrogen from antioxidants. Therefore, DPPH is widely employed to evaluate the free radical scavenging abilities of antioxidants . As illustrated in Figure 3b, FOs showed relatively high DPPH scavenging activity at concentrations ranging from 0.5 to 4.0 mg/ml, and scavenging activity increased as FOs concentration increased. When the concentration at 4.0 mg/ml, the DPPH scavenging activity of FOs was 92.18%, which was similar to BHA (94.49%). It is revealed that FOs are strong free radical scavenging agent at 4.0 mg/ml.
Hydroxyl radicals can induce severe damage to functioning biomolecules in living cells and functional ingredients in food systems by setting off free radical chain reactions (Miao et al., 2020). Thus, hydroxyl radical removal is essential for protecting life systems. The scavenging activity of FOs on hydroxyl radicals is shown in Figure 3c.
The data indicated that the FOs showing a relatively stronger hydroxyl radical scavenging activity in dose-effect relationship than BHA in concentration from 0.5 to 4.0 mg/ml.

The above in vitro antioxidant activities analyses suggested that
FOs can be considered an effective free radical inhibitor. Previous reports have found that the presence of uronic acid in polysaccharides can activate the hydrogen atom of the anomeric carbon, which improves the hydrogen-donating ability of polysaccharides . It is suggested that the free hydroxyl group in polysaccharides may contribute to its antioxidant activity by donating electrons to reduce radicals to more stable forms and/or by directly reacting with the free radicals to terminate the radical chain reaction (Xu et al., 2017).
Additionally, Liu et al. (2017) found that the carboxyl group was the electron-withdrawing group that significantly increased radical scavenging activity of the polysaccharides . Thus, the strong radical scavenging activity of FOs is closely related to its preliminary structure. In this study, FT-IR spectra showed that FOs contained uronic acid, free hydroxyl groups, and carboxyl group. Moreover, the existence of an FA structure provides a phenolic hydroxyl group in FOs, which enhanced its hydrogen atom donation capacity . Furthermore, the proportion of xylose was the highest in the monosaccharide composition of FOs, which has been considered as antioxidant-related monosaccharides .

| Antioxidant activities of FOs in IPEC-J2 cells model
To select appropriate concentrations of FOs for treating IPEC-J2 cells, cell viability was determined by CCK8 assay. As shown in  The results in Figure 5a showed that FOs significantly reduced the fluorescence intensity in IPEC-J2 cells compared with the control (p < .05). The ROS are highly reactive molecules naturally produced in living organisms during the metabolism of oxygen that play an important role in maintaining homeostasis (Ding et al., 2019).
However, overproduction of ROS can cause oxidative stress, which may result in lipid peroxidation, eventually causing cell damage (Kim et al., 2014). As the product of lipid peroxidation, the level of MDA reflects the degree of lipid peroxidation in vivo Mei et al., 2019). In this study, it is observed that MDA levels of the cells treated with FOs at different concentrations (50-400 μg/ml) was significantly decreased (Figure 5b). These results indicated FOs could alleviate lipid peroxidation in IPEC-J2 cells by reducing intracellular ROS levels.
The antioxidant system of living organisms can be divided into enzyme antioxidant defense system and nonenzyme antioxidant defense system. Antioxidant statuses mainly depend on enzymatic antioxidant defense systems. SOD, as an important part of enzyme antioxidant defense system, can remove superoxide anions and protect cells from damage .
GSH-Px is also an important antioxidant enzyme, which can remove hydrogen peroxide and lipid peroxide Yun et al., 2020). CAT is a terminal oxidase that catalyzes the decomposition of hydroxyl radicals (Fang et al., 2020;Mei et al., 2019). As shown in Figure 5c, the SOD activity of cells treated with different concentrations of FOs (100 and 200 μg/ml) was significantly increased (p < .05). Treatment with FOs at 50-200 μg/ml significantly F I G U R E 2 FT-IR spectra of FOs acquired over a frequency range of 4000-400 cm −1 .

F I G U R E 3
Antioxidant activities of FOs: reducing power (a); DPPH radical scavenging activity (b); hydroxyl free radical scavenging activity (c). ABC p <0.05 compared among BHA and FOs; abcd p <0.05 compared among different concentration (p < .05) improved the CAT activity of cells (Figure 5d). After being stimulated with 50-400 μg/ml of FOs, GSH-Px activity of cells was significantly (p < .05) increased (Figure 5e). Similarly, previous report showed that FOs from corn bran significantly increased SOD activity in PC12 cells (Yao et al., 2014). Zhang et al. (2017) reported that FOs enhanced the SOD, GSH-Px, and CAT activities in AAPH-treated HepG2 cells. As the most important and abundant nonenzymatic antioxidant, GSH can directly scavenge hydroxyl radicals and singlet oxygen molecules . Compared with the control, treatment with FOs at concentration of 50-400 μg/ml significantly (p < .05) increased GSH content (Figure 5f). These data suggest that FOs has promoting effects on the antioxidant enzyme secretion and GSH production in IPEC-J2 cells.
Enzymatic antioxidant activities partly depend on the gene expression of antioxidant enzymes. The transcription of antioxidant enzyme genes is typically modulated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) (Hou et al., 2020;Shi et al., 2017). As shown in Figure 6a, treatment with FOs at 200 μg/ ml significantly increased the expression of nuclear Nrf2 (p < .05).
Aside from antioxidant enzymes, phase II detoxification enzymes, such as NAD(P)H: quinone oxidoreductase-1 (NQO-1), heme oxygenase-1 (HO-1), and GSH synthetase were also regulated by Nrf2 signaling pathway (Johnson et al., 2008). The expression of NQO-1 and HO-1 in this study was also markedly (p < .05) upregulated by After being treated with 800 μg/ml of FOs, survival rate was significantly reduced (p < .05). Thus, the concentrations of FOs selected for further research were 50-400 μg/ml. Moreover, this study provided evidence to support the safe use of FOs via zebrafish model.
In this study, we evaluated the effects of FOs on ROS production in zebrafish embryos through detection of an oxidationsensitive fluorescent dye probe (DCF-DA) (Wang, Lee, et al., 2018).
In this study, no effect of FOs treatment on CAT activity in zebrafish embryos was observed (Figure 9a). However, the activity of increased GSH-Px activity (Figure 9c). It suggested that FOs played its antioxidant activity in vivo by increasing the activities of both SOD and GSH-Px. However, there are no previous reports on the effects of FOs in a zebrafish model, it is difficult to make any direct comparisons. Zhang et al. (2015) reported that the activities of SOD, CAT, and GSH-Px in heart, liver, and kidney of rats were increased by FOs treatment, moreover, the increased antioxidant enzymes activities were positively correlated with Nrf2 signaling (Zhang et al., 2015). Experiments were performed in triplicate and data are expressed as the mean ± SE; *p < .05 compared to the control group